In vivo activity of tracheal parasympathetic ganglion cells innervating tracheal smooth muscle

Brain Research. 437 (1987) 157-160

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Elsev,er BRE 22612

nara vmnathetic gancl!ion ceils innervating tracheal smooth muscle

In v i v o ~r_t~iuitv nf trAr.hA:~l

Robert A. Mitchell, Dorothy A. Herbert, David G. Baker and Carol B. Basbaum Departments of Anesthesia, Anatomy, Physiology, and the Cardiovascular Research lnstmae, Umversuv of Cahforma, San Francisco, CA 94143 (U.S A )

(Accepted 18 August 1987) Key words Trachea; Parasympathetic ganghon, Smooth muscle, Airway resistance

In vwo mtracellular recordmg and mtrasomal rejection c,f Lucifer yellow revealed two populations of postganghomc paras.~mpathetic neurons m the tracheal gangha of cats. One consisted of large cells that had an msp,ratory rhythm, had a stgntficant post-spike afterhyperpolanzat~on, and projected to the tracheal smooth muscle. The second consisted of small cells that fired with an expiratory rhythm, had no slgmflcant afterhyperpolanzation, and projected to the mtercartdaginous spaces

In the airway, parasympathetic and sympathetic nerves are believed to interact to regulate the airway smooth muscle tone and mucous secretion 6"9. There have been numerous in vitro neurophysiological studies of the parasympathetic and sympathetic ganglia, but Iew in vivo studies and none in which the end organs innervated by postganglionic parasympathetic nerves have been Identified We have recorded, for the first time in VlVO, intracellular potentials from tracheal parasympathetic ganglion cells and demonstrated that these cells also fire in synchrony with the phrenic nerve. Also, by intracellular iontophoresis of Lucifer yellow, we traced axonal projections of these cells to the tracheal smooth muscle. In cats, anesthetized with a mixture of chlora!oseurethane (40-200 mg/kg), we exposed the trachea through a midline incision and cannulated it, preserving the recurrent laryngeal nerve and the blood supply to the trachea. Phrenic nerve activity was recorded as previously described 45. We created a bilateral pneumothorax and ventilated the cat by a Harvard respiratory pump at a frequency of 6 0 - 7 0 breaths/min with an end-expiratory pressure of 2.5

cm of water. Carbon dioxide ~as added to the inspired gas to maintain the end-expiratory CO~ between 40 and 45 torr. We incised the trachea ventrally along the mldline from the larynx to the tracheal cannula. We mounted the cat in a supine position In a spinal frame and head holder, rotated the tracheal cannula clockwise 90° and stabilized it by a clamp attached to the spinal frame. The incised trachea rostral to the tracheal cannula was rotated another 90 ° and the cut edges were sewn to a lucite platform which was rigidly attached to the spinal frame and illuminated by a fiber optic lamp. Cotton pledgets, saturated with 0.01% Neutral red, a stain for tracheal ganglion cells which does not alter their function s , were placed on the exposed adventitial surface of the trachea for 30 min. The connective tissue overlying the ganglia was removed and the ganglion cells were penetrated with beveled glass electrodes containing 5% Lucifer yellow. Once the membrane potential ~tab:l~ed, we recorded the spontaneous activity of the cells with the end-expiratory pressure at 2.5 cm of H20 and again with the end-expsratory pressure raised to 10 or more cm of H,O. After compleuon of the electrophysiologic studtes, the trachea was fixed

Correspondence R A Mitchell. Rm 1386H S E . Unr~erstty of Cahforma. San Franctsco. CA 94143-0542. U S A

0006-8993/87/$03 50 © 1987 Elsevier Science Pubhshers B.V (Biomedical Division)

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Ftg 1 S~multaneousmtracellular recording of the spontaneous actw~tyof tracheal paras,~mpathet~cganghon umts and phremc nerve actw]ty (Phr) A an respiratory firing (large) parasympatheuc cell (resting membrane potentml -62 mV) B an expiratory firing (small) parasympathetic cell (resting membrane potentm1-58 mV) Note the continuous synaptlc potentmls throughout the respiratory cyclem both these cells and the phasic spike actwlty m the absence of apparent IPSP's.

near the intercartilaginous spaces, although occasionally clusters were located in apposition to the tracheal smooth muscle. These cells fired with an expiratory rhythm or contmuously with increased frequency during expiration. During ]nspiration, small synapt]c potenttals could be observed that failed to ehctt action potentials (Ftg 1B). These small cells were exctted by lung hyperinflation (Ftg 2B), had an average membrane potential of 61 + 8 mV, and had no sigmficant (<3 mV) post-spike afterhyperpolan-

in 4% paraformaldehyde and whole mounts of the region of the gangha were examined by fluorescence microscopy and photographed In 28 cats, we were able to record stable intracellular potentials for up to 30 mln and mject Lucifer yellow mto the ganglion cells We found two types of ganglion cells d]stmgmshed by thexr anatomical and electrophyslological features. Clusters of small ganghon cells (dmmeter 36 _+ 6 Mm, n = 5) were located primarily m the posterolateral tracheal adventitta

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Fig 2 The effect of hypermflatzonof the lung on the flnng pattern of tracheal parasympatheticgangEoncells A inhibition of firing m an msp,ratoryf]nng cell (resting membrane potentm1-61 mV) B" increasedfiring m an expiratory fmng cell (resting membrane potentm1-6.5mV) Phremcnerve actwlty (Phr). and transpulmonarypressure(PTP)

159 zation (Fig IB). When mjected with Lucifer yellow, the cells" axonal projections could be traced into the intercartilaginous spaces toward the nerve plexus about the mucous glands, but the final termination of the axons could not be determined (Fig. 3). A second population of cells of larger diameter (63 +_ 5 gm, n = 27) were located in the adventitm m close apposition to the tracheahs musc!e, near its attachment to the tracheal cartilaginous rings (Fig 3) These cells fired with an insptratory rhythm or continuously with increased frequency during inspiration. Peak frequency was 10-13 Hz, with numerous synaptic potentials in the interspike intervals which failed to reach a threshold sufficlent to evoke an action potential dunng the post-spike afterhyperpolanzatton (Fig 1A). The action potenttals were inhibited by lung hyperinflation, although in many instances, synaptic potentials persisted despite inhibition of phrenic activity (Fig. 2A). These cells had a membrane potential of 52 + 12 mV, and a signifi-

cant afterhyperpolarlzation of 10 _+ 1.2 mV lasting 94 + 18 ms (Fig. 1A) Neither long-lastmg IPSP's nor EPSP's played a role in the rhythmic firing of these cells. Axonal projections extended up to 6 mm from the cell bodies, had numerous vancosities over the last 5 mm, and were m close apposition to the trachealis muscle or projected into it. In previous studies we have shown thai the airway smooth muscle tension is periodic and parallels respiratory activity in the phrentc nerve not only during normal breathing but also during inhibitory and excitatory respiratory reflexes 4-5. In th~s study, we demonstrate that the postganglionlc parasympathetic nerves identffmd as innervatmg the tracheahs muscle also fire with an insptratory rhythm paralleling actwity in the phremc nerve during normal breathing and during refex mMbmon of breathing produced by lung hyperinflatlon, which inhibits the phrenic activity and reduces mrway smooth muscle tone In the one comparable in vivo study of the firing

Fig. 3 Photomicrograph of two types of tracheal parasympathetic ganghon cells, in one cat, intrasomally injected with Lucifer .xello~ after electrophyslologlcalrecordings Two large respiratory finng cells with axons projecting to the tracbeahs smooth muscle (SM) and one small expiratory firing cell with an axon projecting into the mtercartllagmous space Bar = 100 um

160 pattern of parasympathetic ganglion cells, in this case the ciliary gangha, the investigators r e p o r t e d firing rates of 10-15 Hz, synaptic potentials at frequencies in excess of their firing frequencies, and post-spike afterhyperpolarization lasting about 100 ms (ref. 3). This is in marked contrast to the afterhyperpolarizatlon observed in the enteric ganglia which has an onset of 45-80 ms and a duration up to 20 s (ref. 10). There have been no c o m p a r a b l e anatomical or electroph~siological studies in the cat trachea but our observations are similar to those from in vitro studies in the ferret trachea m which two populations of ganghon cells have been described 1'2. The continuous synaptic input in the insplratory firing ganglion cells during inhibition induced by lung hypermflation was similar to the contmuous synaptic input reported by Melnitchenko and Skok 3 in the cil-

1 Baker, D G , McDonald, D M, Basbaum, C B and Mitchell, R A , The architecture of nerves and gangha of the ferret as revealed by acetylchohnesterase hlstochemistry, J Comp Neurol, 246 (1986) 513-526 2 Cameron, A R and Coburn, R F , Electrical and anatomic characteristics o1 the cells of the ferret paratracheal ganghotl, Am J Phystol, 246 (1984) C450-458 3 Meimtchenko. L V and Skok, V.I, Natural electrical activity m mammalian parasympathetic ganghon neurons, Bram Research, 23 (1970) 277-279 4 Mitchell, R A , Herbert, D A and Baker, D G , Insplratory rhythm in atiway smooth muscle tone, J Appl. Phystol, 58 (19~4) 911-920. 5 Richardson, C A , Herbert, D A and Mitchell, R A , Modulation of pulmonary stretch receptors and a~rwav resistance by parasympathetic efferents, J. Appl Physiol., 57 (1984) 1842-1849

tary ganglion during a depressfon of firing, in the absence of IPSP's, produced b y ~.llura,mation of the e s e . Our results are consistent with Skok's observations as well as his view that multiple, synchronous inputs are required to elicit an action potential in p a r a s y m pathetic ganglia 7 and that the modulation of the activity of the parasympathetic ganglit: is achieved by regulation, in the central nervous system, of the n u m ber of active preganglionic fibers lanervating the ganglion cells, rather than by IPSP's acting at the level of the ganglion cells.

We t h a n k S. I k e d a and L. Calonico for their valuable assistance in preparing the figures. This study was supported by N I H G r a n t s HL32572, HL27319, and HL21436

6 Richardson, J B., Nerve supply to the lungs, Am. Rev Resp. Dis, 119 (1979) 785-802 7 Skok, V I , Spontaneous and reflex activities general characteristics In A G Karczamar, K Koketsu and S Nishl (Eds.), Autonomw and Enteric Gangha. Plenum. New York, 1986, pp 425-438 8 Skoogh, B E , Grdlo, M A and Nadel, J A , Neutral red stares gangha in the vagal motor pathway without affecting ganghomc transmission, J. Neurascl Meth., 8 (1983) 33-39 9 Wlddlcombe, J G J , Action potentials m parasympathetic and sympathetic efferent fit~ers to the trachea and lungs of dogs and cats, J Phystol (Lond), 186 (1966) 56-88 10 Wood, J D and Mayer, C J , Intracellular study of electrical actwlty of Auerbach's plexus m guinea pig small intestme, Pflugers Arch, 374 (1978) 265-275